Bioaffinity chromatography has been widely used for sample purification and cleanup,1 chiral separations,2 on-line proteolytic digestion of proteins,3 development of supported biocatalysts,4 and more recently for screening of compound libraries via the frontal affinity chromatography (FAC) method.5,6 The basic premise of FAC is that continuous infusion of a compound will allow for equilibration of the ligand between the free and bound states, where the precise concentration of free ligand is known. In this case, the breakthrough time of the compound will correspond to the affinity of the ligand for the immobilized biomolecule—ligands with higher affinity will break through later.
The detection of compounds eluting from the column can be accomplished using methods such as fluorescence,7 radioactivity,6 or electrospray mass spectrometry.5 The former two methods usually make use of either a labeled library, or use a labeled indicator compound which competes against known unlabelled compounds, getting displaced earlier if a stronger binding ligand is present. However, in each case the methods have limited versatility owing to the need to obtain labeled compounds, and the need for prior knowledge of compounds used in the assay, since no structural information is provided by the detector. Hence, fluorimetric and radiometric methods tend to be useful only for analysis of discrete compounds.
Interfacing of FAC to ESI-MS, on the other hand, has proven to be a very versatile method for screening of compound mixtures.5 Use of MS, and in particular MS/MS detection, provides the opportunity to obtain structural information on a variety of compounds simultaneously. In cases where the identity of compounds in the mixture is known, the analytes can be detected simultaneously and in a quantitative manner using the multiple reaction monitoring (MRM) mode, improving the throughput of the method. While this unique aspect of the FAC/MS technique has been touted as a major advantage for applications such as high-throughput screening of compound mixtures,5,8 there are some potential disadvantages that arise as a result of the use of electrospray ionization for introduction of compounds into the mass spectrometer. For example, obtaining a stable electrospray requires the use of a low ionic strength eluent, which in some cases can be incompatible with maintaining the activity of the proteins immobilized in the column.9 Low ionic strength can also lead to an ineffective double layer, which can cause significant non-selective binding through electrostatic interactions of compounds with the silica column. Furthermore, only one mode of analysis is possible per chromatographic run when using ESI/MS. Finally, high levels of analytes can lead to large ion currents in the electrospray, which can lead to ion suppression.10 
There remains a need for a more compatible and efficient means for detecting compounds eluting from bioaffinity columns.